Franck A. Atienzar

Qualitative assessment of genotoxicity using random amplified polymorphic DNA: Comparison of genomic template stability with key fitness parameters in Daphnia magna exposed to benzo[a]pyrene

A method of DNA profiling using the random amplified polymorphic DNA (RAPD) was used to assess toxicant-induced DNA effects in laboratory populations of Daphnia magna exposed to varying concentrations of the genotoxic hydrocarbon benzo[a]pyrene. These effects, represented by changes in the RAPD profiles, were compared with a number of key ecological fitness parameters (age-specific survival, age-specific fecundity, net reproductive rate, and intrinsic rate of population increase). Not only was the RAPD profiling method shown to be a rapid and reproducible assay of toxicant-induced DNA effects, but the qualitative measure of genomic template stability compared favorably with the traditional indices of fitness. The RAPD profiles, however, exhibited higher sensitivity in detecting toxic effects. The significance of these findings for future ecotoxicological studies is discussed.

Comparison of ultraviolet-induced genotoxicity detected by random amplified polymorphic DNA with chlorophyll fluorescence and growth in a marine macroalgae, Palmaria palmata.

The random amplified polymorphic DNA (RAPD) technique was used to detect DNA damage in the sublittoral macroalgae Palmaria palmata (Rhodophyta) exposed to both ambient and elevated irradiances of UV-B (280-315 nm). To investigate the potential of this method in ecotoxicological assessments, the qualitative and quantitative modifications in RAPD profiles were compared with changes in a number of physiological and fitness parameters. RAPD detectable modifications in DNA profiles were observed in all UV exposed individuals compared with controls. Changes in chlorophyll fluorescence (F(v)/F(m) ratio), in vivo pigment absorptance, thallus growth and RAPD profiles, examined simultaneously, provided a sensitive measure of UV-induced toxicity. In conclusion, the application of the RAPD method in conjunction with other suitable physiological and fitness measurements, may prove to be a valuable tool for investigating the specific effects of genotoxic agents upon marine algal populations. Ultimately, this methodology may allow the ecotoxicological examination of the link between molecular alterations and measurable adverse effects at higher levels of biological organisation.

Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations

The random amplified polymorphic DNA (RAPD) assay and related techniques like the arbitrarily primed polymerase chain reaction (AP-PCR) have been shown to detect genotoxin-induced DNA damage and mutations. The changes occurring in RAPD profiles following genotoxic treatments include variation in band intensity as well as gain or loss of bands. However, the interpretation of the molecular events responsible for differences in the RAPD patterns is not an easy task since different DNA alterations can induce similar type of changes. In this study, we evaluated the effects of a number of DNA alterations on the RAPD profiles. Genomic DNA from different species was digested with restriction enzymes, ultrasonicated, treated with benzo[a]pyrene (B[a]P) diol epoxide (BPDE) and the resulting RAPD profiles were evaluated. In comparison to the enzymatic DNA digestions, sonication caused greater changes in the RAPD patterns and induced a dose-related disappearance of the high molecular weight amplicons. A DNA sample substantially modified with BPDE caused very similar changes but amplicons of low molecular weight were also affected. Appearance of new bands and increase in band intensity were also evident in the RAPD profiles generated by the BPDE-modified DNA. Random mutations occurring in mismatch repair-deficient strains did not cause any changes in the banding patterns whereas a single base change in 10-mer primers produced substantial differences. Finally, further research is required to better understand the potential and limitations of the RAPD assay for the detection of DNA damage and mutations.

4-n-Nonylphenol and 17-β estradiol may induce common DNA effects in developing barnacle larvae

There is a growing concern over the potential effects of environmental endocrine disrupters on both human and wildlife populations. However, to date, minimal research has been conducted to determine the effect of estrogens and xenoestrogens at the DNA level. In this study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the effects on the genomic DNA of barnacle larvae that had been exposed to 17beta-estradiol (E2) and low concentrations of 4-n-nonylphenol (NP). DNA effects include DNA damage as well as mutations and possibly other effects at the DNA level that can be induced by chemical or physical agents that directly and/or indirectly interact with genomic DNA. Not only did exposure to NP and E2 induce changes in RAPD profiles in the exposed barnacle larvae when compared to control patterns, but also, and more importantly, there were similarities in the RAPD modifications in the exposed populations that had been treated to either chemical. We propose that NP and E2 induced some common DNA effects in barnacle larvae and that these specific modifications in RAPD patterns may arise as a consequence of hot spot DNA damage (e.g. DNA adducts) and/or mutations (point mutations or genomic rearrangements). This could help to explain how xenoestrogens mimic the effects produced by natural estrogens. In conclusion, in the field of endocrine disruption, the study of DNA effects induced by estrogens and/or xenoestrogens warrants further investigation. Indeed, changes at the DNA levcl may be the precursors of some of the numerous effects reported at higher levels of biological organisation such as the feminization of males, developmental abnormalities, and infertility.

Use of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations: possible implications of confounding factors.

The aim of this study was to evaluate the potential of the random amplified polymorphic DNA (RAPD) assay to qualitatively detect the kinetics of benzo[a]pyrene (B[Ma]P)-induced DNA effects in the water flea Daphnia magna exposed to 25 and 50 μg l-1 B[a]P for 7 and 6 days, respectively. Mortality was recorded on a daily basis in both experiments, and RAPD analysis was performed on samples collected every day following isolation of genomic DNA. The main changes occurring in RAPD profiles produced by the population of Daphnia magna exposed to 25 and 50μg l-1 B[a]P was a decrease and increase in band intensity, respectively. Most of the changes occurring in the RAPD patterns were likely to be the result of B[a]P-induced DNA damage (B[a]P DNA adducts, oxidized bases, DNA breakages) and/or mutations (point mutations and large rearrangements). In addition, reproducible changes also occurred in the profiles generated by control Daphnia magna. The results lead us to suggest that, in addition to B[a]P-induced DNA damage and mutations, factors such as variation in gene expression, steady levels of genetic alterations and changes in metabolic processes could induce some changes in RAPD patterns. Nevertheless, our data suggest that DNA damage and mutations appear to be the main factors influencing RAPD patterns. This study also emphasizes that unexpected variation in control profiles is not always associated with artefacts.

Application of the arbitrarily primed polymerase chain reaction for the detection of DNA damage

Abstract The technique of arbitrarily primed polymerase chain reaction (AP-PCR) shows potential as a selective and sensitive assay for the detection of xenobiotic-induced DNA damage. Problems, however, may occur in AP-PCR, diminishing its discriminative abilities. These problems include the presence of spurious amplification products in non-template-containing negative control reactions, and a lack of reproducibility amongst amplification patterns. Experiments designed to remove contaminated nucleic acids by ultraviolet (UV) treatment indicated that spurious bands are the result of aberrant primer-induced polymerisation, an event shown to be influenced by the concentration of deoxynucleotide triphosphates (dNTP) present in the reaction mixtures. Optimisation of dNTP concentration from 0.22 to 0.33 m m resulted in clear negative controls and highly reproducible amplification patterns with all DNA templates. As an example of the application of the method, in the present study, the macroalga Palmaria palmata (Rhodophyta) was exposed to UV A and B radiations. The study shows that the AP-PCR method can detect DNA damage and may be useful in detecting such damage following exposure of cells to xenobiotics.